Project Summary Emerging evidence suggests that MrgX1, a G-protein-coupled receptor expressed specifically in the sensory neurons of the dorsal root ganglion (DRG), plays a significant role in modulating nociceptive signaling. However, little progress has been made in the development of small molecule MrgX1 modulators as novel analgesic agents. To this end, we have generated a transgenic mouse line in which human MrgX1 is introduced into the Mrg-cluster?-/- mice (Mrg-/- mice) under the control of mouse MrgC11 promoter. This humanized mouse model, MrgX1;Mrg-/- mice, will serve as an indispensable tool for assessing the preclinical efficacy of MrgX1 modulators devoid of activity against rodent forms of Mrg receptors. Further, submaximal levels of BAM22-derived peptides acting as endogenous MrgX1 orthosteric agonists were detected at the spinal cord dorsal horn during persistent pain while it remains undetectable in the skin. This finding indicates that positive allosteric modulators (PAMs) of MrgX1 may be able to potentiate the effects of the endogenous agonists at the central terminals of sensory neurons without activating peripheral MrgX1. On the basis of this premise, we hypothesize that CNS-penetrant MrgX1 PAMs can serve as novel analgesic agents devoid of itch side effects. This proposal represents one of the new drug discovery research projects that will be undertaken by the Johns Hopkins Drug Discovery Program through collaborative efforts with Johns Hopkins faculty members. By taking full advantage of our extensive expertise and capability in small molecule drug discovery research, we are poised to seize this drug discovery opportunity by executing the following two Specific Aims: (Aim 1) Design and synthesize potent MrgX1 PAMs with desirable ADME profiles; (Aim 2) Test analgesic efficacy of human MrgX1 PAMs in MrgX1;Mrg-/- mice.